CN116808040A - 一种PI3K/mTOR抑制剂NSC781406在制备抗炎药物中的应用 - Google Patents
一种PI3K/mTOR抑制剂NSC781406在制备抗炎药物中的应用 Download PDFInfo
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- CN116808040A CN116808040A CN202310386664.5A CN202310386664A CN116808040A CN 116808040 A CN116808040 A CN 116808040A CN 202310386664 A CN202310386664 A CN 202310386664A CN 116808040 A CN116808040 A CN 116808040A
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Abstract
本发明属于药物治疗学技术领域,具体涉及一种PI3K/mTOR抑制剂NSC781406在制备抗炎药物中的应用。所述NSC781406的结构式如式A所示:实验表明NSC781406能够很好地抑制NO的释放,能够以浓度依赖的方式降低炎症相关蛋白环氧化酶‑2、一氧化氮合酶的表达,以剂量依赖性方式降低大鼠血清中的炎症因子TNF‑α和IL‑1β的生成,表明其有潜力发展成为治疗关节炎的药物。
Description
技术领域
本发明属于药物治疗学技术领域,具体涉及一种PI3K/mTOR抑制剂NSC781406在制备抗炎药物中的应用。
背景技术
炎症是机体抵抗伤害或感染的一种生物保护性反应,是先天免疫的核心组成部分,其局部反应主要表现为红、肿、热、痛和功能障碍。炎症被分为急性炎症和慢性炎症,其中急性炎症表现为炎症的主要症状,而慢性炎症可能在缺乏某些炎症的情况下发生,可导致机体出现严重的病理状况,如自身免疫、糖尿病和癌症等。一般来说,受控的炎症反应是有益的(例如在防止感染方面),但如果调节不当任其发展,可能会导致多种人类疾病的发生与发展,包括炎症性肠病、哮喘、类风湿关节炎、糖尿病和阿尔茨海默病。总之,炎症与多种人类疾病的发生与发展密切相关。炎症通过许多步骤进行调节,其中一个调节步骤是细胞炎症因子网络,这些细胞炎症因子包括一氧化氮(NO),肿瘤坏死因子α(TNF-α)和白介素-1β(IL-1β)。当LPS刺激巨噬细胞后,炎症细胞因子将从免疫细胞中释放,从而导致炎症的产生。同时,在炎症反应中,巨噬细胞激活还会导致炎症介质环氧化酶-2(COX-2)和一氧化氮合酶(iNOS)的过表达。
TLR4(Toll样受体4)是巨噬细胞信号通路中脂多糖(LPS)的细胞膜受体,是主要先天免疫受体介导的炎症信号通路的一个组成部分,它可以识别病原体相关的分子模式,并激活转录因子以产生各种促炎细胞因子,清除入侵的病原体。目前它已经被证明与多种炎症性疾病密切相关,可以调节人体系统的免疫稳态,被认为是最有影响力的靶点之一。髓样分化蛋白2(MD2)是TLR4的辅助蛋白,抑制TLR4/MD-2的激活可以有效降低TLR4相关炎症细胞因子的表达,从而减轻炎症反应。当LPS刺激TLR4/MD-2复合体时,启动一系列信号级联,导致NF-κB和MAPK信号通路的激活,以及包括NO、诱导型一氧化氮合酶(iNOS)在内的下游细胞促炎因子的诱导产生和表达,如IL-1β、IL-6、TNF-α和环氧合酶-2(COX-2)等。
目前用于治疗炎症的经典药物为非甾体类抗炎药物,如阿司匹林,吲哚美辛等。但这些药物引起的一些不良反应,包括胃和肾毒性,限制了其长期使用。因此,开发更有效的、具有较低副作用的抗炎药物变得尤为重要。
发明内容
为了解决上述技术问题,本发明提供一种PI3K/mTOR抑制剂NSC781406和/或NSC781406在药理学上容许的盐在制备抗炎药物中的应用,所述NSC781406的结构式如式(A)所示:
优选的,所述NSC781406在药理学上容许的盐包括NSC781406与无机酸、有机酸、碱金属、碱土金属或碱性氨基酸中任一种成的盐;所述无机酸为盐酸、硝酸、硫酸、磷酸、氢溴酸中的任一种,所述有机酸为马来酸、富马酸、酒石酸、乳酸、柠檬酸、乙酸、甲磺酸、对甲苯磺酸,己二酸,棕榈酸,单宁酸中的任一种,所述碱金属为锂,钠、钾中的任一种;所述碱土金属为钙、镁中的任一种;所述碱性氨基酸为赖氨酸。
本发明还提供一种用于治疗炎症的药物,其含有药学上有效剂量的NSC781406和/或NSC781406在药理学上容许的盐,所述NSC781406的结构式如式(A)所示:
优选的,所述NSC781406在药理学上容许的盐包括NSC781406与无机酸、有机酸、碱金属、碱土金属或碱性氨基酸中任一种成的盐;所述无机酸为盐酸、硝酸、硫酸、磷酸、氢溴酸中的任一种,所述有机酸为马来酸、富马酸、酒石酸、乳酸、柠檬酸、乙酸、甲磺酸、对甲苯磺酸,己二酸,棕榈酸,单宁酸中的任一种,所述碱金属为锂,钠、钾中的任一种;所述碱土金属为钙、镁中的任一种;所述碱性氨基酸为赖氨酸。
优选的,所述药物还包含有药学上可接受的载体。
优选的,所述药学上可接受的载体包括赋形剂、稳定剂、抗氧化剂、着色剂、稀释剂、缓释剂中的一种或几种功能的辅料;如淀粉、脂类、蜡、糊精、蔗糖、乳糖、微晶纤维素、明胶、柠檬酸、无机盐类、羟丙基甲基纤维素、羟乙基纤维素等。
优选的,所述药物为注射剂、片剂、丸剂、胶囊剂、悬浮剂或乳剂中的任意一种。
本发明的有益效果为:
喹啉类衍生物NSC781406是一种被广泛认知的PI3K(磷脂酰肌醇3-激酶)和mTOR(哺乳动物雷帕霉素靶蛋白)抑制剂,PI3K-mTOR通路在导致细胞增殖、存活和血管生成的众多血液瘤或实体瘤中发挥着关键作用,抑制PI3K-mTOR信号通路是抗肿瘤的重要手段。NSC781406作为新型高效的PI3K和mTOR双重抑制剂,在60种癌细胞中的平均GI50为65nM,其中4种癌细胞系的GI50小于10nM,此外,在肝癌细胞异种移植模型中也表现出有效的肿瘤生长抑制作用,长久以来都是作为靶向性肿瘤药物使用。
本发明通过细胞实验证明,喹啉类衍生物NSC781406能够很好地抑制NO的释放,表明该类化合物可以作为抗炎药物应用。
本发明通过实验证明,喹啉类衍生物NSC781406能够以浓度依赖的方式降低炎症相关蛋白环氧化酶-2(COX-2)、一氧化氮合酶(iNOS)的表达,以剂量依赖性方式降低大鼠血清中的炎症因子TNF-α和IL-1β的生成,表明其有潜力发展成为治疗关节炎的药物。
附图说明
图1为实施例1中NSC781406虚拟筛选和活性测试的技术路线。
图2是实施例4中NSC781406对细胞RAW264.7上清中IL-1β和TNF-α的影响,*p<0.01,**p<0.001与LPS刺激的细胞相比。
图3是实施例5中NSC781406对炎症相关蛋白质COX-2和iNOS表达的影响。
图4为实施例5中NSC781406对炎症相关蛋白质COX-2和iNOS表达影响的柱状图,###p<0.0001与对照组相比,**p<0.001,***p<0.0001与LPS刺激的细胞相比。
图5为实施例6中NSC781406对LPS诱导的NF-κB信号通路的抑制作用,图6为实施例6中NSC781406对LPS诱导的MAPK信号通路的抑制作用,图5中a和图6中a均为蛋白质免疫印迹分析,图5中b和图6中b均为蛋白质的定量数据;###p<0.0001与对照组相比,*p<0.01,**p<0.001,***p<0.0001与LPS刺激的细胞相比。
图7为实施例7中各处理组大鼠足部特征结果。
图8为实施例7中NSC781406对佐剂诱导的关节炎模型大鼠的影响,其中A、B、C分别对应体重、肿胀度和关节炎指数。
图9为H&E染色观察实施例7中NSC781406对AIA模型大鼠关节病理组织的影响(放大倍数:×10)。
图10为实施例7中NSC781406对佐剂诱导关节炎模型大鼠炎症因子IL-1β(图10中A)、TNF-α(图10中B)的抑制结果,###p<0.0001与对照组相比,**p<0.001,***p<0.0001与LPS刺激的细胞相比。
具体实施方式
除非另有说明,本文中所使用的术语均具有本领域技术人员常规理解的含义。
下面结合实施例对本发明的技术方案做出更为具体的说明。实验使用到的喹啉类衍生物NSC781406通过MCE官网购买获得(CAS No.:1676893-24-5)。
实施例1
基于受体结构的药效团模型(SBP)的虚拟筛选:
TLR4/MD2(PDB编码:3FXI)的晶体结构从Protein Data Bank(PDB,https://www.rcsb.org/)官网上获得。使用Discovery Studio 2017完成蛋白质的制备,然后利用其共晶配体构建结合位点,交互生成协议实现程序,并根据球体内的活性位点残基生成HBA、HBD和HY特征集。随后在特征交互图中进行编辑、分类、聚类和排列,最终得到了基于受体结构的药效团模型(SBP)。选择商业数据库Chemdiv(350,000个化合物)和MCE(7231个化合物)进行虚拟筛选。通过配体制备模块制备了总共357231个化合物,并通过使用完全最小化模块使能量最小化。在经过类药五原则和ADMET的初步过滤后,SBP筛选出的化合物选择一百个化合物进行购买,化合物参考实施例2表1。喹啉类衍生物NSC781406虚拟筛选和活性测试的技术路线,如图1所示。
实施例2
CCK-8实验检测细胞毒性实验
将腹腔巨噬细胞RAW264.7细胞按照5000/孔的密度接种到96孔板中,细胞贴壁后除去培养基,用PBS冲洗细胞两次。用补充有10%FBS和含药(实施例1筛选化合物,含喹啉类衍生物NSC781406,20μM)的新鲜培养基37℃,5%CO2条件下培养24小时,每孔100μL。温育后96孔板中每孔再加入CCK-8溶液10μL,并将96板再温育2小时。然后将96孔板放在酶标仪中450nm处测吸光度值(OD450值)。吲哚美辛Indomethacin作为阳性对照。通过计算得到用药组相对于对照组的细胞增值比率。空白组为不加细胞只加培养基,对照组为不加药细胞组。
细胞存活率=(实验组OD450-空白组OD450)/(对照组OD450-空白组OD450)。
然后用SPSS软件计算IC50。结果以三次实验的平均值±SD表示。结果如表1所示。
表1筛选化合物对RAW264.7细胞存活率的影响
从表1可看出,有12个化合物在20μM下细胞存活率低于50%,而其余88个化合物(含喹啉类衍生物NSC781406)在20μM下的细胞存活率高于50%。为了排除接下来的抑制NO生产能力实验中细胞毒性的干扰。选择这些化合物(含喹啉类衍生物NSC781406)进行接下来的抑制NO生产能力测试。
实施例3
细胞NO抑制活性实验
在LPS诱导的RAW264.7细胞模型中,通过测试化合物抑制NO释放情况来评估其抗炎能力。
RAW264.7细胞培养在DMEM培养基(10%胎牛血清和青霉素100U/mL、链霉素100U/mL)。将RAW264.7细胞以每孔6×104个接种于48孔板中培养24h(37℃,5%CO2)。弃掉旧培养基,加入预先配制好的含药培养基(实施例2筛选88个化合物,含NSC781406)预处理1小时,然后每孔加入30μL LPS(1μg/ml)共孵育24小时。取50μL细胞培养液上清于96孔板中,采用ELISA方法,每孔依次加入50μL Griess assay agent I与Griess assay agent II混合,室温孵育10分钟,在540nm下用多功能酶标仪测定吸光度。结果如表2所示。
表2筛选化合物抑制LPS诱导的RAW264.7细胞中NO产生的能力
从表2可看出,GS-444217、MK-2461和NSC781406在10μM下对NO的抑制能力高于90%,且明显高于阳性药(吲哚美辛Indomethacin),接下来选择该三个化合物测定该化合物的IC50。
IC50定义为LPS刺激RAW264.7细胞产生50%NO时化合物的浓度,结果以三次实验的平均值±SD表示。
表3化合物对NO产生的抑制作用
从表3可看出,NSC781406表现出最好的NO抑制活性(IC50=1.89±0.41μM),并明显优于阳性药。实验表明本发明提供的喹啉类衍生物NSC781406在一定程度上能够抑制炎症因子NO的释放,表明喹啉类衍生物NSC781406有潜力发展成为抗炎药物。
实施例4
炎症因子IL-1β和TNF-α的释放实验
在LPS诱导的RAW264.7细胞模型中,测试化合物NSC781406抑制炎症因子IL-1β和TNF-α的释放情况。
用不同浓度的NSC781406(10μM,5μM,2.5μM,1.25μM,0.625μM)一起孵育细胞,使用ELISA试剂盒(基因美,武汉)按照说明书方法测定化合物NSC781406对细胞上清IL-1β和TNF-α的影响,结果见图2。
从图2可以看出,NSC7814068能以剂量依赖性方式降低细胞上清中的炎症因子IL-1β和TNF-α的释放。经计算,NSC7814068对IL-1β的IC50在1.54±0.43μM,对TNF-α的IC50在2.81±0.38μM。
实施例5
蛋白质印迹(Western Blot)实验检测NSC7814068抑制COX-2和iNOS的表达能力
将RAW264.7细胞与不同浓度的NSC781406(1μM、3μM和10μM)和阳性药物Bay11-7082(5μM)共同孵育1小时,然后用LPS刺激细胞24小时。通过蛋白质印迹分析NSC781406对环氧化酶-2(COX-2)、一氧化氮合酶(iNOS)的表达的抑制能力,进一步评估化合物的抗炎作用,结果见图3、4。
图3为NSC781406对炎症相关蛋白表达的影响图,可以看出随着NSC781406浓度的降低,炎症蛋白COX-2和iNOS的表达量明显升高。图4为显示至少三个独立实验的平均值±SD(n=3)结果的柱状图,可以看出,与模型组(LPS)相比,NSC781406以浓度依赖性方式降低COX-2和iNOS的表达。表明NSC781406可以抑制炎症蛋白(COX-2、iNOS)的表达。
实施例6
蛋白质印迹(Western Blot)实验检测NSC7814068抑制LPS诱导的NF-κB/MAPK信号通路的激活实验
将RAW264.7细胞与不同浓度的NSC781406(1μM、3μM和10μM)共同孵育1小时,然后用LPS刺激细胞24小时。通过蛋白质印迹分析NSC781406抑制NF-κB/MAPK相关通路蛋白的表达作用,结果见图5、图6。
图5为NSC781406抑制RAW 264.7细胞NF-κB信号通路图,左图a为蛋白质免疫印迹分析,右图b为蛋白质的定量数据。可以看出NSC781406抑制IκB的磷酸化和降解来阻断IκB激酶的激活,此外,NSC781406还能以浓度依赖性的方式抑制P65的磷酸化,阻止NF-κB P65从细胞质向细胞核的转位。
图6为NSC781406抑制RAW 264.7细胞中的MAPK信号通路图左图a为蛋白质免疫印迹分析,右图b为蛋白质的定量数据。从图中可以看出,NSC781406(1,3和10μM)也能抑制LPS诱导的细胞P38、JNK和ERK的磷酸化。
实施例7
佐剂性关节炎(Adjuvant arthritis,AIA)实验
炎症反应中,巨噬细胞表面的膜识别受体被脂多糖(LPS)刺激后激活炎症通路,因此巨噬细胞常被用作体外模型研究炎症。巨噬细胞激活会导致环氧化酶-2(COX-2)、一氧化氮合酶(iNOS)过表达。RA相关动物实验模型的构建多为实验室标准情况下的发病,所以本实验尝试从病证结合动物模型进行研究RA。RA病证结合动物模型建立在RA模型的基础上,RA模型中佐剂型关节炎(AIA)模型应用较广泛,其起病急,有自愈性,可以模拟RA急性发病与缓解的病程特点,在这些基础上,本实验选择佐剂性关节炎模型。
1)实验动物:
雌性SD大鼠(180-220g),安徽医科大学实验中心提供。大鼠在温湿度可控(23℃~25℃,40%~60%,12h)的标准条件下饲养。
2)佐剂性关节炎诱导及实验处理:
将50只大鼠随机分为5组,模型组给大鼠的左后足注射0.1mL完全弗氏佐剂(FCA)引起炎症,正常组给大鼠在同一部位注射等量生理盐水。在FCA注射10天后,正常组和模型组采用0.5%羧甲基纤维素钠(CMC-Na)溶液灌胃14天,药物组采用具有不同浓度的化合物NSC781406(30mg/kg、10mg/kg)灌胃14天,阳性对照组采用吲哚美辛(10mg/kg)灌胃14天。
各处理组的大鼠足部特征结果见图7、图8。可以看出,使用NSC781406(10和30mg/kg)和阳性药物(10mg/kg)用于治疗大鼠佐剂性关节炎(AIA)模型,与模型组相比,药物组以浓度依赖性方式减轻了脚的肿胀(图7和图8B),并且当给药剂量≥10mg/kg时,给药组体重得到改善(图8A),在第24天,30mg/kg的NSC781406显著降低了关节炎指数(图8C)。
3)H&E染色
取大鼠踝关节炎症,4%多聚甲醛溶液固定,5%甲酸脱钙,石蜡包埋。切片用H&E染色。光学显微镜下观察组织病理学变化。
图9可以看出,与正常组比较,模型组大鼠滑膜细胞增殖明显,炎症细胞浸润明显,而经NSC781406治疗组大鼠滑膜细胞增殖和炎症细胞浸润明显减轻。
4)IL-1β和TNF-α含量的体内测定
麻醉各处理组大鼠后,从心脏动脉采血,静置30分钟后以4℃在3000r/min离心10分钟收集血清,然后采用ELISA方法测定血清中IL-1β和TNF-α的水平。实验结果见图10。
图10可以看出,大鼠血清中的炎症因子IL-1β、TNF-α在正常组、模型组的生成均显著增加,但NSC781406能以剂量依赖性方式降低大鼠血清中的炎症因子IL-1β、TNF-α的生成。
上述实验表明NSC781406有潜力发展成为治疗关节炎的药物。
以上仅为本发明的较佳实用例而已,并不用以限制本发明创造;尽管参照前述实施方式对本发明进行了详细的说明,本领域的普通技术人员应当理解:凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明创造的保护范围之内。
Claims (7)
1.一种PI3K/mTOR抑制剂NSC781406和/或NSC781406在药理学上容许的盐在制备抗炎药物中的应用,所述NSC781406的结构式如式(A)所示:
2.如权利要求1所述的应用,其特征在于,所述NSC781406在药理学上容许的盐包括NSC781406与无机酸、有机酸、碱金属、碱土金属或碱性氨基酸中任一种成的盐;所述无机酸为盐酸、硝酸、硫酸、磷酸、氢溴酸中的任一种,所述有机酸为马来酸、富马酸、酒石酸、乳酸、柠檬酸、乙酸、甲磺酸、对甲苯磺酸,己二酸,棕榈酸,单宁酸中的任一种,所述碱金属为锂,钠、钾中的任一种;所述碱土金属为钙、镁中的任一种;所述碱性氨基酸为赖氨酸。
3.一种用于治疗炎症的药物,其含有药学上有效剂量的NSC781406和/或NSC781406在药理学上容许的盐,所述NSC781406的结构式如式(A)所示:
4.如权利要求3所述的药物,其特征在于,所述NSC781406在药理学上容许的盐包括NSC781406与无机酸、有机酸、碱金属、碱土金属或碱性氨基酸中任一种成的盐;所述无机酸为盐酸、硝酸、硫酸、磷酸、氢溴酸中的任一种,所述有机酸为马来酸、富马酸、酒石酸、乳酸、柠檬酸、乙酸、甲磺酸、对甲苯磺酸,己二酸,棕榈酸,单宁酸中的任一种,所述碱金属为锂,钠、钾中的任一种;所述碱土金属为钙、镁中的任一种;所述碱性氨基酸为赖氨酸。
5.如权利要求3所述的药物,其特征在于,所述药物还包含有药学上可接受的载体。
6.如权利要求4所述的药物,其特征在于,所述药学上可接受的载体包括赋形剂、稳定剂、抗氧化剂、着色剂、稀释剂、缓释剂中的一种或几种功能的载体。
7.如权利要求4所述的药物,其特征在于,所述药物为注射剂、片剂、丸剂、胶囊剂、悬浮剂或乳剂中的任意一种。
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